Abstract
Active elimination of apoptotic cells is very important for maintaining homeostasis of early embryos. Recent observations on mouse blastocysts freshly isolated from healthy dams have shown that the majority of incidentally occurring apoptotic cells is eliminated by neighbouring embryonic cells. Some apoptotic cells escape phagocytosis, but the frequency of such processes usually does not exceed 10%. The aim of the current study was to evaluate whether the non-professional embryonic phagocytes can respond to experimentally induced increase in apoptosis by increasing the frequency of efferocytosis and whether their activity can be decreased by selective inhibition of specific component of efferoctosis machinery. Experiments were performed in vitro on cultured mouse blastocysts with a differentiated trophectoderm and inner cell mass and on the human trophoblast cell line Ac-1M88. Samples were assessed using fluorescence immunostaining: Apoptotic cells (TUNEL) internalised within the cytoplasm of non-professional embryonic phagocytes (phalloidin T membrane staining) were considered ingested; apoptotic cells co-localised with acidified phagosomes (LysoTracker) were considered digested. First, we tested the ability of embryonic phagocytes to respond to elevated incidence of apoptosis induced by actinomycin D (4 nM). The results showed that the increase in apoptosis was accompanied by a significant elevation of the phagocytosis and digestion of dead cells in both mouse blastocysts and human trophoblast cells. We then assessed the effect of selective inhibition of lysosomal acidification in embryonic phagocytes using a specific V-ATPase inhibitor bafilomycin A1. The results showed that the inhibitor at 0.1 and 0.2 nM was able to negatively affect the execution of both initiative and terminal phases of efferocytosis in mouse blastocysts, although the decrease was not as profound as expected. When compared to mouse trophectoderm cells, human hybrid cells displayed a very low sensitivity to bafilomycin A1. Higher concentrations of bafilomycin A1 had a more harmful impact on overall cell viability than on digestive activity. The results show that the ability of non-professional embryonic phagocytes to successfully execute all stages of efferocytosis is not limited by the frequency of apoptosis and is preserved even at elevated rates of the apoptotic process. The effectiveness of embryonic phagocytes can be partially decreased by selective inhibition of lysosomal acidification conducted via V-ATPase.